The present invention relates to the area of chemotherapeutic agents and, more particularly, relates to certain substituted caprolactams, pharmaceutical compositions comprising said caprolactams, a method of treating tumors, the use of said caprolactams in the chemotherapy of tumors, and a process for preparing said compounds.
Cancer is a serious health problem throughout the world. As a result, an extensive number of research endeavors has been undertaken in an effort to develop therapies appropriate to the treatment and alleviation of cancer in humans.
In the chemotherapeutic area, research has been conducted to develop anti-tumor agents effective against various types of cancer. Oftentimes, anti-tumor agents which have been developed and found effective against cancer cells are, unfortunately, also toxic to normal cells. This toxicity manifests itself in weight loss, nausea, vomiting, hair loss, fatigue, itching, hallucinations, loss of appetite, etc., upon administration of the anti-tumor agent to a patient in need of cancer chemotherapy.
Furthermore, conventionally used chemotherapeutic agents do not have the effectiveness desired or are not as broadly effective against different types of cancers as desired. As a result, a great need exists for chemotherapeutic agents which are not only more effective against all types of cancer, but which have a higher degree of selectivity for killing cancer cells with no or minimal effect on normal healthy cells. In addition, highly effective and selective anti-tumor agents, in particular, against cancers of the colon, bladder, prostate, stomach, pancreas, breast, lung, liver, brain, testis, ovary, cervix, skin, vulva and small intestine are desired. Moreover, anti-tumor activity against colon, breast, lung and prostate cancers as well as melanomas are particularly desired because of the lack of any particular effective therapy at the present time.
The present invention provides new anti-tumor agents which are effective against a variety of cancer cells. More particularly, the present invention relates to certain substituted caprolactams which exhibit a high degree of selectivity in killing cancer cells. The essence of the instant invention is the finding that certain substituted caprolactams are useful in treating tumors.
The invention relates to caprolactams of formula I: 
where
R1 is (C1-6)alkyl or (C3-6)cycloalkyl;
R2 is hydrogen or (C1-6)alkyl;
X is (C1-12) alkylene; (C2-12) alkenylene; or (C2-12) alkynylene; 
m is 0 or 1; and
R3 is (C3-8)cycloalkyl; or an aromatic ring system selected from II, III, IV and V:
xe2x80x83where
R4 is hydrogen, chloro, or methoxy;
R5 is hydrogen, chloro, (C1-18)alkyl or (C1-18)alkoxy; and Z is oxygen, sulfur, Nxe2x80x94H, or Nxe2x80x94CH3;
or a pharmaceutically acceptable acid addition salt thereof, where possible.
Preferred compounds of formula I are those where
R1 is (C1-6) alkyl;
R2 is hydrogen or (C1-4) alkyl;
X is (C1-6) alkylene or (C2-6) alkenylene;
m is 0 or 1; and
R3 is (C3-8)cycloalkyl; or an aromatic ring system selected from II, III, IV and V where
R4 is hydrogen, chloro, or methoxy;
R5 is hydrogen, chloro, (C1-18)alkyl or (C1-18)alkoxy; and Z is oxygen, sulfur, Nxe2x80x94H, or Nxe2x80x94CN3;
or a pharmaceutically acceptable acid addition salt thereof, where possible.
More preferred compounds are those of formula I where
R1 is i-propyl or t-butyl;
R2 is hydrogen or methyl;
m is 0 or 1;
X is (C1-6) alkylene; and
R3 is (C5-7)cycloalkyl; or an aromatic ring system selected from IIa and V: 
xe2x80x83where
R4xe2x80x2 is in the meta position and is hydrogen or chloro; and
R5xe2x80x2 is in the para position and is hydrogen, chloro, (C1-18)alkyl or (C1-18)alkoxy;
or a pharmaceutically acceptable acid addition salt thereof, where possible.
Even more preferred compounds are those of formula I where
R1 is i-propyl or t-butyl;
R2 is hydrogen or methyl;
m is 0 or 1;
X is methylene or ethylene; and
R3 is (C5-7)cycloalkyl, phenyl, 3,4-dichlorophenyl, 4-methoxyphenyl, 4-n-decylphenyl, 4-n-decyloxyphenyl or 3-pyridyl;
with the proviso that when m is 0, R3 is (C5-7)cycloalkyl, 4-n-decylphenyl or 4-n-decyloxyphenyl;
or a pharmaceutically acceptable acid addition salt thereof, where possible.
In another embodiment, the instant invention provides pharmaceutical compositions, especially for the treatment of tumors in warm-blooded animals, comprising a pharmaceutically acceptable carrier or diluent and an antitumorally effective dose of a compound of formula I above, preferably 3,4,5-trihydroxy-2-methoxy-8,8-dimethyl-N-[hexahydro-2-oxo-6-(cyclohexylcarbonyl)oxy-2H-azepin-3-yl]non-6-enamide or 3,4,5-trihydroxy-2-methoxy-8,8-dimethyl-N-[hexahydro-2-oxo-6-(1-oxo-3-phenylpropoxy)-2H-azepin-3-yl]non-6-enamide, or a pharmaceutically acceptable acid addition salt thereof, where possible.
In still another embodiment, the instant invention provides a method for treating tumors comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound of formula I above, or a pharmaceutically acceptable acid addition salt thereof, where possible.
In another embodiment, the instant invention relates to the use of a compound of formula I or of a pharmaceutically acceptable salt of such a compound for the preparation of a pharmaceutical composition for use in the chemotherapy of tumors.
Furthermore, the instant invention relates to the use of a compound of formula I or of a pharmaceutically acceptable salt of such a compound for the chemotherapy of tumors.
In the above definitions: 1) the alkyl groups containing 1 to 6 carbon atoms are either straight or branched chain, of which examples of the latter include isopropyl, isobutyl, t-butyl, isopentyl, neopentyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 1,1,2,2-tetramethylethyl; and 2) the alkyl and alkoxy groups containing 1 to 18 carbon atoms are either straight or branched chain.
The term xe2x80x9c(C1-2) alkylenexe2x80x9d as used herein refers to a straight or branched chain divalent group consisting solely of carbon and hydrogen and having from 1 to 12 carbon atoms. Examples of xe2x80x9calkylenexe2x80x9d groups include methylene, ethylene, propylene, butylene, pentylene, 3-methypentylene, etc.
The term xe2x80x9c(C2-12) alkenylenexe2x80x9d as used herein refers to a straight or branched chain divalent group consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond, and having from 2 to 12 carbon atoms. Examples of xe2x80x9calkenylenexe2x80x9d groups include ethenylene, propenylene, butenylene, pentenylene, 3-methylpentenylene, etc.
The term xe2x80x9c(C2-12) alkynylenexe2x80x9d as used herein refers to a straight or branched chain divalent group consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond, and having from 2 to 12 carbon atoms. Examples of xe2x80x9calkynylenexe2x80x9d groups include acetylene, propynylene, butynylene, pentynylene, 3-methylpentynylene, etc.
The acid addition salts of the compounds of formula I may be those of pharmaceutically acceptable organic or inorganic acids. Although the preferred acid addition salts are those of hydrochloric and methanesulfonic acid, salts of sulfuric, phosphoric, citric, fumaric, maleic, benzoic, benzenesulfonic, succinic, tartaric, lactic and acetic acid may also be utilized.
The caprolactams of formula I may be prepared as depicted below: 
where each R1, R2, X, m and R3 is as defined above.
As to the individual steps, Step A involves the acylation of an aminocaprolactam of formula VI with a lactone compound of formula VII to obtain a diamide compound of formula VIII. The acylation is conducted in a polar, organic solvent, preferably a protic polar solvent such as isopropanol, at a temperature slightly below or at the reflux temperature of the solvent employed for a period of between 4 and 48 hours.
Alternatively, the acylation of an aminocaprolactam of formula VI, or an acid addition salt thereof, with the lactone compound of formula VII in Step A may be carried out with in the presence of: 1) a weak base, preferably a carboxylate salt such as sodium 2-ethylhexanoate, and 2) a polar, organic solvent, preferably an ether such as tetrahydrofuran, at a temperature of between 0xc2x0 C. and 50xc2x0 C., preferably at 25xc2x0 C., for a period of between 1 hour and 7 days, preferably for 20 hours.
Step B concerns the hydrolysis of the 1,3-dioxane group common to a diamide compound of formula VIII, to obtain a substituted caprolactam compound of formula I. The hydrolysis is typically carried out by dissolving the diamide in a mixture of solvents consisting of 1) a protic acid, preferably an organic acid such as trifluoroacetic acid, 2) a protic solvent, preferably water, and 3) an inert organic solvent, preferably a cyclic ether such as tetrahydrofuran, at a temperature of between 0xc2x0 C. and 25xc2x0 C. for a period of between 5 minutes and 2 hours.
Alternatively, the diamide compounds of formula VIII may be prepared according to the following 3-step reaction scheme: 
where X, m and R3 and each R1 and R2 are as defined above, and R6 is an alcohol protective group. Preferably, R6 is a silyl group such as tert-butyldimethylsilyl.
As to the individual steps, Step 1 involves the acylation of an aminocaprolactam of formula IX with a lactone compound of formula VII to obtain a diamide compound of formula X. The acylation is conducted in the presence of a base, preferably an alkylamine base such as diisopropylethylamine, and a polar, organic solvent, preferably a protic polar solvent such as isopropanol, at a temperature slightly below or at the reflux temperature of the solvent employed for a period of between 4 and 48 hours.
Step 2 concerns the hydrolysis of the group R6 common to a diamide compound of formula X to obtain a hydroxycaprolactam compound of formula XI. The hydrolysis is typically carried out in the presence of fluoride, preferably a fluoride salt such as tetrabutylammonium fluoride, and an inert organic solvent, preferably a cyclic ether such as tetrahydrofuran, at a temperature of between 0xc2x0 C. and 25xc2x0 C. for a period of between 5 minutes and 2 hours.
Step 3 concerns the acylation of a hydroxycaprolactam compound of formula XI by reacting it with an acid chloride of formula R3(X)mCOCl where R3, X and m are as defined above, to obtain a diamide compound of formula VIII. The acylation is conducted in the presence of a base, preferably an alkylamine base such as triethylamine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
Alternatively, the acylation of a hydroxycaprolactam compound of formula XI in Step 3 may be carried out with a carboxylic acid of formula R3(X)mCO2H where R3, X and m are as defined above, in the presence of a carboxylic acid coupling reagent, preferably a diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a suitable activating agent common to diimide coupling reactions, preferably a substituted pyridine such a 4-dimethylaminopyridine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
The aminocaprolactam compounds of formula VI may be prepared as depicted below: 
where each R6, R2, X, m and R3 is as defined above, and each R7 is a carbonyl-containing group. Preferably, R7 is alkoxycarbonyl such as t-butyloxycarbonyl.
As to the individual steps, Step 1a involves the cyclization of hydroxylysine (or any salt or hydrate preparation thereof) XII to obtain hydroxycyclolysine XIII. The cyclization is typically carried out in the presence of a coupling reagent, preferably a diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a suitable activating agent common to diimide coupling reactions, preferably an N-hydroxy compound such as 1-hydroxybenztriazole hydrate, and a base, preferably an alkylamine base such as triethylamine, and a polar organic solvent, preferably an amide such as N,N-dimethylformamide, at a temperature of between 0xc2x0 C. and 40xc2x0 C. for a period of between 12 and 72 hours.
Step 2a involves the N-acylation of hydroxycyclolysine XIII to obtain an N-acylhydroxycyclolysine compound of formula XIV. The acylating agent is typically an acid chloride or an anhydride. When R7 is t-butyloxycarbonyl, the acylating agent is di-tert-butyldicarbonate. The reaction is carried out in the presence of a base, preferably an alkylamine base such as triethylamine, and a polar organic solvent, preferably an amide such as N,N-dimethylformamide, at a temperature of between 0xc2x0 C. and 40xc2x0 C. for a period of between 1 and 24 hours.
Step 3a involves the O-silylation of an N-acylhydroxycyclolysine compound of formula XIV to obtain a silyl ether compound of formula XV. The silylating agent is typically a silyl chloride or trifluoromethanesulfonate. When R6 is tert-butyldimethylsilyl, the silylating agent is tert-butyldimethylsilylchloride. The reaction is carried out in the presence of a base, preferably a mild base such as imidazole, and a polar organic solvent, preferably an amide such as N,N-dimethylformamide, at a temperature of between 0xc2x0 C. and 40xc2x0 C. for a period of between 1 and 24 hours.
Step 4a involves the N-alkylation of a silyl ether compound of formula XV with an alkyl (defined as R2 above) halide or sulfonate to obtain an N-alkyl caprolactam compound of formula XVI. The alkylation is conducted in the presence of a strong base, preferably an alkali metal amide such as sodium bis(trimethylsilyl)amide, and an inert organic solvent, preferably a cyclic ether such as tetrahydrofuran, at a temperature of between xe2x88x92100xc2x0 C. and 25xc2x0 C. for a period of between 5 minutes and 2 hours.
Step 5a concerns the hydrolysis of the group R6 common to an N-alkyl caprolactam compound of formula XVI, to obtain a hydroxycaprolactam compound of formula XVII. The hydrolysis is typically carried out in the presence of fluoride, preferably a fluoride salt such as tetrabutylammonium fluoride, and an inert organic solvent, preferably a cyclic ether such as tetrahydrofuran, at a temperature of between 0xc2x0 C. and 25xc2x0 C. for a period of between 5 minutes and 2 hours.
Step 6a concerns the acylation of a hydroxycaprolactam compound of formula XVII to obtain an ester compound of formula XVIII by reacting it with an acid chloride of formula R3(X)mCOCl where R3, X and m are as defined above, in the presence of a base, preferably an alkylamine base such as triethylamine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
Alternatively, the acylation of a hydroxycaprolactam compound of formula XVII in Step 6a may be carried out with a carboxylic acid of formula R3(X)mCO2H where R3, X and m are as defined above, in the presence of a carboxylic acid coupling reagent, preferably a diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a suitable activating agent common to diimide coupling reactions, preferably a substituted pyridine such a 4-dimethylaminopyridine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
Step 7a concerns the hydrolysis of the group R7 on an ester compound of formula XVIII to obtain an aminocaprolactam compound of formula VI. The hydrolysis is typically carried out in the presence of a protic acid, preferably an organic acid such as trifluoroacetic acid, hydrogen or a silyl halide, preferably a silyl iodide such as trimethylsilyl iodide, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x92100xc2x0 C. and 25xc2x0 C. for a period of between 1 minute and 2 hours.
The aminocaprolactam compounds of formula VIa may be prepared as depicted below: 
where each R7, X, m, and R3 is as defined above.
As to the individual steps, Step 1b concerns the acylation of a hydroxycaprolactam compound of formula XIV to obtain an ester compound of formula XVIIIa by reacting it with an acid chloride of formula R3(X)mCOCl where R3, X and m are as defined above, in the presence of a base, preferably an alkylamine base such as triethylamine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
Alternatively, the acylation of a hydroxycaprolactam compound of formula XIV in Step 1b may be carried out with a carboxylic acid of formula R3(X)mCO2H where R3, X and m are as defined above, in the presence of a carboxylic acid coupling reagent, preferably a diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a suitable activating agent common to diimide coupling reactions, preferably a substituted pyridine such a 4-dimethylaminopyridine, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x9278xc2x0 C. and 25xc2x0 C. for a period of between 1 and 24 hours.
Step 2b concerns the hydrolysis of the group R7 on an ester compound of formula XVIIIa to obtain an aminocaprolactam compound of formula VIa. The hydrolysis is typically carried out in the presence of an protic acid, preferably an organic acid such as trifluoroacetic acid, hydrogen or a silyl halide, preferably a silyl iodide such as trimethylsilyl iodide, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x92100xc2x0 C. and 25xc2x0 C. for a period of between 1 minute and 12 hours.
The aminocaprolactam compounds of formula IXa may be prepared as depicted below: 
where R7 and each R6 are as defined above. The reaction concerns the hydrolysis of the group R7 on an ester compound of formula XV to obtain an aminocaprolactam compound of formula IXa. The hydrolysis is typically carried out in the presence of a protic acid, preferably an organic acid such as trifluoroacetic acid, hydrogen or a silyl halide, preferably a silyl iodide such as trimethylsilyl iodide, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between xe2x88x92100xc2x0 C. and 25xc2x0 C. for a period of between 1 minute and 2 hours.
The lactone compounds of formula VII may be prepared as depicted below: 
where R1 is as defined above.
As to the individual steps, Step 1c involves the diketalization of polyhydroxylated lactone of formula XIX with acetone to obtain bis(acetonide) XX. The diketalization is conducted in acetone as solvent using a catalyst such as iodine at a temperature of between 0xc2x0 C. and the reflux temperature for a period of between 2 and 48 hours.
Step 2c involves the methylation of bis(acetonide) XX with a methylating agent such as methyl iodide to obtain the methyl ether XXI. The methylation is conducted in the presence of water and a base, preferably a metal oxide such as silver oxide, and an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between 0xc2x0 C. and the reflux temperature for a period of between 12 hours and 7 days.
Step 3c involves the hydrolysis of methyl ether XXI to obtain the dihydroxy compound of formula XXII. The hydrolysis is conducted in the presence of water and a protic acid, preferably a carboxylic acid such as acetic acid, at a temperature of between 5xc2x0 C. and 35 C for a period of between 1 and 24 hours.
Step 4c involves the oxidative cleavage of dihydroxy compound XXII to obtain the aldehyde XXIII. The reaction is conducted in the presence of an oxidant, preferably a periodate salt such as sodium periodate, in a protic solvent, preferably an alkanol such as methanol, at a temperature of between 0xc2x0 C. and 25xc2x0 C. for a period of between 10 minutes and 4 hours.
Step 5c involves the olefination of aldehyde XXIII to obtain a lactone compound of formula VII. The olefination is conducted in the presence of an organometallic compound, preferably an organochromium compound such as the transient species generated from chromium(II)chloride and a diiodoalkane (defined as R1CHl2where R1 is as defined above), in the presence of a solvent mixture consisting of 1) a polar organic solvent, preferably an amide such as N,N-dimethylformamide, and 2) an inert organic solvent, preferably a cyclic ether such as tetrahydrofuran, at a temperature of between xe2x88x9280xc2x0 C. and 25xc2x0 C. for a period of between 5 minutes and 4 hours.
Although the product of each reaction described above may, if desired, be purified by conventional techniques such as chromatography or recrystallization (if a solid), the crude product of one reaction is advantageously employed in the following reaction without purification.
As is evident to those skilled in the art, the substituted caprolactam compounds of formula I contain asymmetric carbon atoms. It should be understood, therefore, that the individual stereoisomers are contemplated as being included within the scope of this invention.
As indicated above, certain of the compounds of formula I form pharmaceutically acceptable acid addition salts. For example, the free base of a compound of formula I can be reacted with hydrochloric acid to form the corresponding hydrochloride salt form, whereas reacting the free base of the compound of formula I with methanesulfonic acid forms the corresponding mesylate salt form. All pharmaceutically acceptable addition salt forms of the compounds of formula I are intended to be embraced by the scope of this invention.
In a further embodiment, the present invention relates to a process for preparing a caprolactam compound of formula I which comprises, in a first step, acylating an amino caprolactam compound of formula VI 
with a lactone compound of formula VII 
in the presence of a polar, organic solvent to obtain a diamide compound of formula VIII 
where each of R1, R2, X, m and R3 are as defined above and, in a second step, hydrolyzing the diamide compound obtained in the first step by dissolving it in a mixture of solvents to obtain the desired caprolactam compound of formula I. Preferably, the acylation in the first step is conducted in isopropanol at a temperature slightly below or at the reflux temperature of the isopropanol, whereas the hydrolysis in the second step is conducted in a mixture consisting of a protic, organic acid, a protic solvent and an inert, organic solvent, more preferably a mixture consisting of trifluoroacetic acid, water and tetrahydrofuran.
As indicated above, all of the compounds of formula I, and their corresponding pharmaceutically acceptable acid addition salts, are anti-tumor agents and are, therefore, useful in inhibiting the growth of various lymphomas, sarcomas, carcinomas, myelomas, and leukemia cell lines. The anti-tumor activity of the compounds of formula I may be demonstrated employing the Anchorage Dependent Growth Monolayer Assay (ADGMA) which measures the growth inhibitory effects of test compounds on proliferation of adherent cell monolayers. This assay was adapted from the 60 cell line assay used by the National Cancer Institute (NCl) with the following modifications: 1) cell lines representative for the important tumor types, viz., MDA-MB-435 breast, A549 non-small cell lung, HCT-116 colon and PC-3 prostate carcinomas were utilized; and 2) a tetrazolium derivative, viz., MTS, was utilized to determine cell density.
The ADGMA compares the number of viable cells following a 3-day exposure to a test compound relative to a number of cells present at the time the test compound was added. Cell viability is measured using a tetrazolium derivative, viz., 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) that is metabolically reduced in the presence of an electron coupling agent (PMS; phenazine methosulfate) by viable cells to a water-soluble formazan derivative. The absorbance at 490 nm (A490) of the formazan derivative is proportional to the number of viable cells. The IC50 for a test compound is the concentration of compound required to reduce the final cell number to 50% of the final control cell number. If cell proliferation is inhibited, the assay further defines compounds as cytostatic (cell number after 3-day compound incubation  greater than cell number at time of compound addition) or cytotoxic (cell number after 3-day compound incubation  less than cell number at time of compound addition).
The MDA-MB-435 breast carcinoma line was obtained from the American Type Culture Collection (ATCC) and used between passages 4-20 following thawing. MDA-MB-435 breast carcinoma was maintained and plated in DME/F12 medium containing 10% fetal bovine serum, 15 mM HEPES (pH=7.4), 100 units/mL penicillin, and 100 xcexcg/mL streptomycin.
The A549 non-small cell lung, HCT-116 colon, and PC-3 prostate carcinoma lines were obtained from the American Type Culture Collection (ATCC) and used between passages 4-20 following thawing. A549, HCT-116 and PC-3 cells were maintained in RPMI 1640 containing 5% FBS, 5 mg/mL insulin, 5 mg/mL transferrin, 5 mg/mL selenous acid, 1 nM xcex2-estradiol, 1 nM testosterone, 100 units/mL penicillin and 100 ug/mL streptomycin.
Cell lines are trypsinized and counted using a Coulter counter to determine plating densities. Cells are then plated in their respective maintenance media (100 xcexcL/well) in 96 well plates at the following densities: MDA-MB-435, 3,000 cells/well; A549 and HCT-116, 700 cells/well; are PC-3, 2500 cells/well. The number of cells plates as determined in preliminary experiments, results in cell densities of 75-90% of confluency by 4 days after plating. Initial cell densities, assayed one day after plating, are roughly 0.15-0.20 absorbance units greater than the media blank. Ninety-six well plates are seeded on day 0 and the test compounds are added on day 1. A control plate is created for each cell line that receives media only in row A and cells in row B. One day following plating, test compounds are added (in a final volume of 100 xcexcL) to the test plates. Control plates receive 10 xcexcL MTS mixture (prepared fresh on day of addition to cell plates at a ratio of 10 xcexcL of a 0.92 mg/mL solution of PMS to a 190 xcexcL of a 2 mg/mL solution of MTS) and 100 xcexcL media. A490 of control plates is read 4 h after MTS addition to determine initial cell density values for each cell line. Three days after addition of test compound, 10 xcexcL/well of MTS mixture is added to the test plates and A490 is read 4 h later. A490 values for wells containing cells are corrected for media absorbance, then normalized to initial density readings to determine percent net growth. IC50 values are determined from graphs of percent net growth as a function of compound concentration. Percent net growth is calculated as (Cell+Drug A490xe2x88x92Initial A490/Cell+Drug Vehicle A490xe2x88x92Initial A490).
The following IC50 values (averagexc2x1S.D.) in xcexcM were obtained:
The anti-tumor activity of the compounds of formula I may further be demonstrated employing the athymic (T cell deficient) nude mouse model which has been and remains the standard for drug discovery and development in preclinical oncology. Utilizing this model, one can measure the ability of test compounds to inhibit the growth of human tumor xenografts growing subcutaneously (s.c.) in athymic nude mice. The histologic tumor types employed were MDA-MB-435 breast, A549 non-small cell lung, HCT-116 colon and PC-3 prostate carcinomas.
MDA-MB-435 breast carcinoma xenograft model: Briefly, 3 million cells were implanted s.c. into the right flank of athymic (nu/nu) mice, and were allowed to grow until a mass of approximately 30 mm3 was established. The test compounds are administered three times per week intravenously (i.v.) for three weeks in 5% dextrose, 10%DMSO in water. The test compounds are administered in dose-response fashion in order to evaluate and document the full potential range of activity (efficacy and toxicity) for a given compound. Positive controls are carried out with doxorubicin administered 3 times per week i.v.
A549 non-small cell lung carcinoma xenograft model: Briefly, 10 million cells were implanted s.c. into the right flank of athymic (nu/nu) mice, and were allowed to grow until a mass of approximately 120 mm3 was established. The test compounds are administered three times per week intravenously (i.v.) for three weeks in 5% dextrose, 10%DMSO in water. The test compounds are administered in dose-response fashion in order to evaluate and document the full potential range of activity (efficacy and toxicity) for a given compound. Positive controls are carried out with mitomycin C administered 3 times per week intraperitioneally (i.p.).
HCT-116 colon carcinoma xenograft model: Briefly, 3.5 million cells were implanted s.c. into the right flank of athymic (nu/nu) mice, and were allowed to grow until a mass of approximately 45 mm3 was established. The test compounds are administered three times per week intravenously (i.v.) for three weeks in 5% dextrose, 10%DMSO in water. The test compounds are administered in dose-response fashion in order to evaluate and document the full potential range of activity (efficacy and toxicity) for a given compound. Positive controls are carried out with 5-fluorouracil administered 1 time per week i.v.
PC-3 prostate carcinoma xenograft model: Briefly, approximately 20 mm3 fragments were implanted s.c. into the right flank of athymic (nu/nu) mice, and were allowed to grow until a mass of approximately 100 mm3 was established. The test compounds are administered three times per week intravenously (i.v.) for three weeks in 5% dextrose, 10%DMSO in water. The test compounds are administered in dose-response fashion in order to evaluate and document the full potential range of activity (efficacy and toxicity) for a given compound. Positive controls are carried out with paclitaxel (dissolved in 12.5% cremophore/12.5% ethanol/0.9% saline solution) administered i.v. daily for the first five days of the study.
Toxicity was monitored by recording average group body weights twice weekly, and by daily observation of general health. Efficacy was monitored by taking measurements of tumor length, width, and depth weekly using digital calipers coupled to automated data collectors. Mean tumor volume (MTV) at initiation of therapy was subtracted from final MTV in order to express the actual tumor growth during treatment (xcex94MTV). Anti-tumor activity was expressed as %T/C (xcex94MTV of treated group÷xcex94MTV of control groupxc3x97100). Regressions were calculated using the formula: (T/T0xe2x88x921)xc3x97100%, where T is the tumor volume for the treatment group at the end of the experiment, and T0 is the tumor volume at the beginning of the experiment. Statistical significance was evaluated using a one-tailed Student""s t-test (p less than 0.05).
The following results were obtained for compounds of Ex.1-Ex.12 tested against MDA-MB-435 tumor xenografts 3xc3x97/week for 3 weeks:
The following results were obtained for compounds of Ex.""s 1, 2, 4, and 5 tested against A549 tumor xenografts 3xc3x97/week for 3 weeks:
The following results were obtained for compounds of Ex.""s 1, 2, 4, and 5 tested against HCT-116 tumor xenografts 3xc3x97/week for 3 weeks:
The following results were obtained for compounds of Ex""s 1 and 4 tested against PC-3 tumor xenografts 3xc3x97/week for 3 weeks:
The precise dosage of the compounds of formula I to be employed for inhibiting tumors depends upon several factors including the host, the nature and the severity of the condition being treated, the mode of administration and the particular compound employed. However, in general, satisfactory inhibition of tumors is achieved when a compound of formula I is administered parenterally, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally, e.g., orally, preferably intravenously or orally, more preferably intravenously at a daily dosage of 1-300 mg/kg body weight or, for most larger primates, a daily dosage of 50-5000, preferably 500-3000 mg. A preferred intravenous daily dosage is 1-75 mg/kg body weight or, for most larger primates, a daily dosage of 50-1500 mg. A typical intravenous dosage is 20 mg/kg, three to five times a week.
Usually, a small dose is administered initially and the dosage is gradually increased until the optimal dosage for the host under treatment is determined. The upper limit of dosage is that imposed by side effects and can be determined by trial for the host being treated.
The compounds of formula I may be combined with one or more pharmaceutically acceptable carriers and, optionally, one or more other conventional pharmaceutical adjuvants and administered enterally, e.g. orally, in the form of tablets, capsules, caplets, etc. or parenterally, e.g., intraperitoneally or intravenously, in the form of sterile injectable solutions or suspensions. The enteral and parenteral compositions may be prepared by conventional means.
The infusion solutions according to the present invention are preferably sterile. This may be readily accomplished, e.g. by filtration through sterile filtration membranes. Aseptic formation of any composition in liquid form, the aseptic filling of vials and/or combining a pharmaceutical composition of the present invention with a suitable diluent under aseptic conditions are well known to the skilled addressee.
The compounds of formula I may be formulated into enteral and parenteral pharmaceutical compositions containing an amount of the active substance that is effective for inhibiting tumors, such compositions in unit dosage form and such compositions comprising a pharmaceutically acceptable carrier.